Whole mount RNA in situ hybridization
藥品配製 1.胚胎培養液 :14 mM NaCl :0.54 mM KCl :0.026 mM Na2HPO4 :0.1 mM CaCl2 :0.1 mM MgSO4．7H2O 2.色素抑制劑(phenylthiocarbamide)(1-phyeny-z-thiourea, PTU) :stock solution 2mM : ↓10x 稀釋 :working solution 0.2mM 3. PBS(per liter) :8 mg NaCl :2.88mg Na2HPO4 :0.4mg KCl :0.48mg KH2PO4, pH7.4 4. 4% paraformaldehyde/PBS solution（保存期限：1星期） :取paraformaldehyde(存於4℃中)，溶於PBS solution中，使其濃度為4%(v/v)。於68℃水溶槽中靜置，待溶解後，保存於-20℃中。 5. proteinase K stock solution 6. yeast tRNA 10mg/ml 7. PBT solution: 0.1% tween20 in PBS 8. HYB-: (pH=5.0)以citric acid (1M or 2M均可)調整pH，設總體積為500ml時： :60% pormamide (300ml) ::4x SSC (取100ml 20x SSC) ::0.1% Tween (取500μl) Tween怕光，要包錫箔紙。 溶於DEPC-H2O中 9. HYB+: (pH=5.0)以citric acid調pH值。設體積為250ml :60% pormamide (150ml) ::4x SSC (取50ml 20x SSC) ::0.1% Tween (取250μl) Tween怕光，要包錫箔紙。 ::50μg/ml Heparin (取12.5mg) ::500μg/ml Yeast tRNA (取10mg/ml yeast tRNA 12.5ml) 溶於DEPC-H2O中 預留空間調pH值 10. maleic acid 1M pH7.5 11. citric acid 1M 12. Tris-HCl 1M pH=9.5(以HCl調整) :配100ml：取12.11g tris，加入80ml 3`H2O，以HCl調整pH，把總體積加到100ml 13. MgCl2 1.0M 取20.33g加三次水至100ml 14. NaCl 1.5M 取8.766g加三次水至100ml 15. NaCl 1.0M 取5.844g加三次水至100ml 16. 100mM Maleic acid (3x) 取 300mM maleic acid 100ml 17. 150mM NaCl(10x) 取1.5M NaCl 30ml 18. 19.0.1% Tween 20　　取300μl (將17和18混合後，加三次水至總體積為300ml) 20. 4x blocking reagent ：取2g blocking reagent 粉末(存於4℃冰箱)加50ml上列混合液。Blocking reagent不易溶解，配置好後經過高壓滅菌，即可溶解完全。 21. 20x SSC (per liter) :175.3mg NaCl :88.2mg NaOAc :adjust to pH=7.0(可省略) :→滅菌 22. NTMT solution :0.1M NaCl :0.1M Tris-HCl(用HCl調pH值) :50mM MgCl2 :0.1% Tween 20 探針置備｜RIBOPROBE PREP 1. plasmid DNA linearized: :取10μg DNA，以restriction enzyme作用。 :經過phenol, chloroform, 酒精沉澱 :用DEPC-ddH2O將體積調至10μl 2. 取1μg purified template DNA 以下步驟在冰上進行 :Using Roche kit 1 175 025 :(1)先加入適當體積的DEPC-ddH2O，使最終體積為20μl :(2)加入template DNA 1μg :(3)加入0.5μl (RNase inhibition) :(4)加入2μl (DIG/flurescin labble mix) :(5)加入2μl (10x buffer) :(6)加入2μl (SP6 orT7) :mix gently, spin down 3. 37℃ 2小時 4. 加入2μl DNase1，37℃ 20分鐘。 5. 加入formamide 80μl。取1μl跑電泳。 6. 保存於-20℃。 實驗步驟 1.收集卵，置於胚胎培養液中，培養於28.5℃備用。 2.第14hr後加色素抑制劑PTU(ex:27ml 3’水加3ml PTU(2mM)最後濃度為0.2mM) 3.將embryo置於tube中(最多30顆)加入4% paraformaldehyde/ PBS solution，置於4℃中12~16小時，或置於室溫2hr 4.　剝除卵膜 5.　脫水 Dehydration :200μl 25% methanol + 75% PBT 5min :200μl 50% methanol + 50% PBT 5min :200μl 75% methanol + 25% PBT 5min :200μl 100% methanol 10min×3 脫水後可保存於-20℃中，約可保存6個月 6.　Re-hydration :200μl 75% methanol + 25% PBT 5min :200μl 50% methanol + 50% PBT 5min :200μl 25% methanol + 75% PBT 5min :200μl 1x PBT 5min×4 第4次用以PBT配製的proteinase K處理 7.proteinase K :incubate embryos in 200μl proteinase K at room temperature(RT) *Stop reaction with 200μl PBT *re-fix embryos in 200μl 4% paraformaldehyde/PBS for 20min at RT *wash embryos with 200μl PBT for 5min 3times. 8.Pre-hybrydization :preheat HYB- to 60℃ :incubate embryos in 200μl preheated HYB- at 65℃ for 5min :pre-hybridize embryos in 200μl HYB+ at 65℃for 3hrs 9.'''Heat RNA probe'''-HYB+ (each 200μl HYB+ containing 20～100ng RNA probe) at 68℃ for 10min, 快速置於冰上。 Add 500μl RNA probe- into embryos. Incubate embryos at 65℃overnight. 10.Post-hybridization washing wash embryos with preheated solution :75% HYB-/25% 2x SSCT 65℃ 10min :50% HYB-/25% 2x SSCT 65℃ 10min :25% HYB-/25% 2x SSCT 65℃ 10min :2×SSCT 65℃ 10min :0.2×SSCT 70℃ 45min×2 11.Absorption(anti-dig antibody reaction) :rinse the embryo with 200μl maleic acid buffer(100mM maleic acid, 150mM NaCl, 0.1%Tween 20)then decade. :add 500μl 1xBlocking reagent and stand at RT for 3 hrs. :decade blocking reagent and add 500μl antibody (anti-DIG-AP antibody, Roche) incubate at 4℃ overnight :wash embryo with 200μl maleic acid buffer for 30min ×4times. 12. 呈色Coloring :加入新配製的NTMT buffer, 5min, 3times, at RT. (NBT/BCIP在NTMT的pH下最易呈色。故先以NTMT改變embryo的pH值。) :500μl NBT/BCIP containing buffer, 1-2hrs, at RT :stop reaction with quick rinses in PBT for several times. 或10min, 4 times. :wash embryos with methanol for 20min 1~3times at RT (洗去不必要的背景。) :wash embryo with PBS and store in PBS at 4℃. 13.拍照 討論｜Discussion category:實驗